MODULE 5 - Putting Microbes to Work in the Environment

Question 1

why do biofilms have to be on wet surfaces?

Answer 1

they have to have a moisture content so the bacteria can survive

Question 2

what are some nutrients and environmental factors that form gradients?

Answer 2

water

pH

temp.

oxygen

pressure

radiation

Question 3

how can a bacteria survive starvation through morphological changes?

Answer 3

endospores (metabolically dormant, resistant to heat)

nucleoid condensation (nucleoid associated proteins bend DNA so it can condense, stress the bacteria and they switch to starvation mode where gene transcription changed, so stress response pathways activated and growth slowed)

Question 4

how can bacteria survive starvation with starvation proteins?

Answer 4

transpeptidases which cause peptidoglycan cross-linking and thickening of cell wall

chaperones which prevent denaturation and help renature damaged proteins

Question 5

why are starved cells hard to kill?

Answer 5

the can survive for years

can become more virulent

Question 6

how does formation of persister cells occur?

Answer 6

small subset of cells which are spontaneously dormant (non-growing) even with nutrients available

starvation triggers persister cells through nutrient depletion leading to stress adaptation

formation of persister cell occurs in exponential phase

Question 7

why are persister cells important?

Answer 7

they can’t be treated with antibiotics

they don’t harbour antibiotic ressitance genes and instead express antibiotic tolerance phenotype

they are tolerant to the immune system and so are all together hard to eradicate

Question 8

what is the difference between antibiotic resistance and antibiotic tolerance?

Answer 8

resistance based on genetic mutation on chromosome, while tolerance means the bacteria overcomes the antibiotic but isn’t resistant to it

Question 9

what’s worse than having persister cells infecting you?

Answer 9

having persister cells in a biofilm infecting you

biofilm formation can also enhance persister cell formation by stressing bacteria

Question 10

what are the stages of biofilm development?

Answer 10

reversible attachment

irreversible attachment (EPS starts to be secreted)

maturation

maturation and dispersion

Question 11

where in the biofilm are you likely to find bacteria with a tolerant phenotype?

Answer 11

in the deeper levels where they are under more stress (O2 and nutrient limitations)

when bacteria don’t grow that well they might randomly start mutating chromosome, if antibiotic treatment happening they especially can develop R genes

Question 12

can biofilms have multiple type of microorganism in them?

Answer 12

yes

they can be individual or polymicrobial (e.g. bacteria, fungi)

Question 13

in what stage of biofilm formation are bacteria most vulnerable to eradication?

Answer 13

during reversible attachment

during irreversible attachment bacteria much more tolerant to antibiotics, immune system and starvation and generally cannot be eradicated by antibiotics alone

Question 14

what is extracellular polymeric substances and what does it do?

Answer 14

major component of a biofilm (50-95% of dry weight)

chemical composition may vary between different strains/enviros but is primarily polysaccharide

protects from desiccation, antibiotics, toxins, immune cells

maintains integrity of biofilm and binds essential nutrients (making local rich environment)

Question 15

what are the advantages of biofilms?

Answer 15

physical attachment (in moving enviros like river, GI tract, bloodstream)

this beneficial as allows it to stay in one place, substrate may provide nutrients, extracellular enzymes that solubilise don’t get diluted quickly, nutrients may be higher in biofilm than enviro

Question 16

what is the medical significance of biofilms?

Answer 16

delay wound healing

increase risk of infection (chronic, polymicrobial)

protects from body’s immune response (inflam response may induce biofilm formation)

providing nutrients in form of exudate (from dead immune cells)

damages healing tissue

Question 17

how common are biofilm bacteria involved in chronic (non-healing) wounds?

Answer 17

biofilm bacteria in 60-90% chronic wounds

these wounds are stuck in inflammatory phase of healing and cannot progress further

often not easily realised and bacteria feed on host response

Question 18

how can host immune response worse biofilm formation in wound?

Answer 18

neutrophils and macrophages come to wound to release proteases (e.g. matrix metalloproteinases (MMPs)) as these degrade dead tissue and extracellular matrix proteins

problem is when too many pathogens in wound more and more neutrophils come and produce more metalloproteases which provides more nutrients for bacteria by degrading dead tissue

in normal physiological conditions MMP levels controlled

Question 19

what are matrix metalloproteinases (MMPs)?

Answer 19

proteases which require metal as co-factor for catalytic activity to occur

factor in abnormal healing in chronic wounds due to over production and increased protease activity

unbalanced activity facilitates colonisation and proliferation in chronic wounds

bacteria also secrete them so in bad wound lots of tissue being degraded thus lots of nutrients for bacteria

Question 20

how are biofilms a problem for our teeth?

Answer 20

dental plaque is a biofilm and formation damages tooth and causes receding gums and bad breath

causes periodontal disease, dental caries

biofilms on teeth a symptom

flossing/brushing regularly prevents this

Question 21

what are some biofilm infections that form from objects?

Answer 21

catheter insertion causing UTIs, vascular disease (bloodstream)

catheter contaminated often through caregivers hand

piercings, tattoos and brandings

Question 22

how can we prevent biofilm formation on wounds?

Answer 22

regularly clean and debride wound tissue, remove biofilm material, necrotic tissue, foreign material in order to prevent biofilm maturation

sometimes difficult as biofilms might be attached to healthy tissue

most effective in early stages

Question 23

how can we manage biofilm once it has formed in a wound?

Answer 23

increase frequency of debridement

cannot completely remove biofilm

once removed prevent re-establishment, otherwise this can happen within 24h

Question 24

how do we treat bacterial biofilms once they are formed in wounds?

Answer 24

biofilms become resistant to most antimicrobials within 48-96hrs

clinical management requires complete removal of infected area

if not possible, attacking on regular schedule may force biofilm detachment, make bacteria susceptible for treatment and host defences HOWEVER can cause systemic infection e.g. sepsis, bacteraemia which are more virulent infections

Question 25

what are the stages of biofilm development?

Answer 25

initial attachment phase primary colonisation phase climax community phase

Question 26

what influences adhesion of the first few bacteria in a biofilm?

Answer 26

linked to environmental cues and quorum sensing they also produce biosurfactants to break down water content in an environment

Question 27

what are the surfaces bacteria adhere to before forming biofilm?

Answer 27

attachment often occurs on rough surfaces e.g. a crack to get stuck in if on smooth surface could be due to flow boundary layer where flow pushes bacteria against surfaces

Question 28

describe the initial attachment phase?

Answer 28

microbes attach to a surface or themselves attachment helped with flagella, filaments (help bacteria recognise surface structures), fimbriae (little charged hairs which are attracted to certain surfaces) and pili mobile bacteria in aqueous phase become loosely attached via electrostatic or hydrophobic interaction w surface

Question 29

describe the primary colonisation phase?

Answer 29

permanent attachment and symbiotic community production of EPS and extracellular DNA which is the foundation of the biofilm interaction with substrate which is mediated by production of extracellular polymers by colonising bacteria once attached to surface active growth begins, micrcolonies form on substrate, further growth continues to cover entire surface

Question 30

describe the climax community phase of biofilm development?

Answer 30

develops within seven days, growth continues until steady state once mature biofilm may contain bacteria that can't even attach to substrate or survive initial nutritional restrictions but other bacteria clutched up (polymicrobial)

Question 31

what is biofouling?

Answer 31

accumulation (usually a biofilm) of bacteria on a surface they are not wanted on e.g. industrial equipment, ship

Question 32

what is economies of scale?

Answer 32

ideally you spend lots on equipment at first and then over time produce more product and so it pays off the investment so you have to find sweet spot where investment pays off as equipment lasts longer

Question 33

what are common places in industry where biofilm formation is a problem?

Answer 33

power plants, air conditioning, food processing, oil refining biofilms cause major production disruptions

Question 34

what are some industrial examples of financial losses due to biofilms?

Answer 34

food industries lose large production due to contamination fouling of pipes leads to friction and plant failure through corrosion and lesser flow heat exchanger surfaces lead to inefficient heat exchange cleaning requires closing of entire plant and scrubbing of plumbing which decreases production

Question 35

how are biofilms a major problem for the dairy industry?

Answer 35

milk highly perishable as has high nutrient content so easy for microbes to grow sterile in udder cells biofilms form at air-liquid interface and are usually bacillus species (floating biofilms)

Question 36

outline the bacillus genus and how it is bad for dairy industry?

Answer 36

gram-positive rod shaped highly detrimental as cause dairy spoilage and illness associated with animal udders and then spread through diary production systems prod heat resistant endospores allowing persistence through pasteurisation and then biofilm formation

Question 37

what are some biofilm components produced by bacillus?

Answer 37

exopolysaccharides amyloid-like fibres (non-soluble proteins) - soluble proteins which fold into insoluble fibres allowing further resistance

Question 38

what are some common methods of cleaning equipment for biofilm control and what are some problems with this?

Answer 38

use cheap chemical agents such as chlorine, NaOH, acid chlorination is most common methods however problem is some microbes resistant and chlorine also not good enough to eliminate biofilms as leaves EPS matrix intact

Question 39

what are some examples of damage caused to at home equipment from biofilms?

Answer 39

rubber seals in washing machine and dishwasher

Question 40

what is the dairy plant cleaning in place (CIP) procedure?

Answer 40

cold water and acid wash immediately after milking hot water and NaOH wash to remove adhered residues at least twice a week cold water acid wash after each alkali wash to remove minerals, kill remaining bacteria and neutralise alkali solution final wash w detergent

Question 41

how acids destroy cells?

Answer 41

denature proteins, extremely reactive and break apart chemical structures, upon exposure cells die but don't disintegrate

Question 42

how do alkalis destroy cells?

Answer 42

accept protons (i.e. rip them the fuck off other molecules), found in cleaners and bleach burns from alkalis way worse than acid burns as they liquify biological material

Question 43

how does biofouling cause problems in NZ involving marine pests and disease?

Answer 43

underside of bots prone to bacteria attachment and biofilm formation this can spread harmful bacteria to prevent this must clean hull of ship, also can use antifouling coating vessels must show evidence of biofouling management

Question 44

outline some key forms of biofouling management?

Answer 44

clean niche areas e.g. protrusions, recesses in deck, unpainted parts of hull maintenance procedures e.g. paint w antifouling paints, marine growth prevention systems (e.g. approved chemicals), steam blow-out pipes

Question 45

how are certain surfaces anti-fouling?

Answer 45

reagents/dispersants such as repelling agents, slippery surface coatings and nanoscale surface topologies

Question 46

how can biocides prevent fouling on the hulls of boats?

Answer 46

chemical removal of biofouling and can be incorporates into antifouling coatings specifically kills bacteria, fungi and algae e.g. tributyltin (TBT)

Question 47

what is tributyltin (TBT)?

Answer 47

a common biocide which is now banned component of paint and slowly released into environment very poisonous to marine life e.g. shellfish highest concentrations in harbours where boats with this coating moored

Question 48

do antifouling coatings kill microorganisms?

Answer 48

nah they just prevent them from attachment

Question 49

what are the three main classes of antifouling coatings?

Answer 49

hydrophobic coating hydrophilic coating polymer coating

Question 50

what are hydrophobic coatings?

Answer 50

repel water low friction and surface energy smooth surface problem is no mechanical strength and only short term stability (so have to be reapplied frequently) e.g. silicone or umbrellas/rain jackets

Question 51

what are hydrophilic coatings?

Answer 51

attract water highly hydrated zwitterions composed of glycine betaine get saturated w water molecules and repel organisms, enzymes and EPS low friction and work better then hydrophobic surfaces in regards to biofilm formation however not commercially available

Question 52

what are polymer coatings?

Answer 52

poly(ethylene glycol) - PEG hydrophilic but water soluble so difficult to use as a coating in aqueous enviro research on mussel adhesive proteins (MAPs) prod by blue mussel and adding this to PEG to make it more adhesive

Question 53

what are some antifouling examples from nature?

Answer 53

bacteria don't attach to shark skin, butterfly wings, seaweed so there are antimicrobial antifouling compounds in nature we haven't discovered which could be mimicked for engineering purposes

Question 54

what are some coatings we could add to our teeth to prevent oral biofilms?

Answer 54

Microparticles w quorum sensing inhibitors to block biofilm formation calcium-binding polymer coated microparticles could allow sustained release of QS inhibitors

Question 55

how did oil get dispersed far as fuck when deepwater horizon happened?

Answer 55

grease-cutting soaps such as dispersants break oil into small bits and so it merged with deep sea water and sunk it also spread as dragged down by marine snow and wind/currents pushed it far away

Question 56

when they couldn't find like a quarter of the oil spilt from deepwater horizon, what happened to it?

Answer 56

hydrocarbon-degrading bacteria in the sediments mineralised it to CO2 or immobilised it as biomass

Question 57

what is bioremediation?

Answer 57

engineered process using microbes to break down environmental pollutants from a spillage microbes involved are often extremophiles as they can tolerate high levels of organic solvents

Question 58

what are hydrocarbonoclastic bacteria?

Answer 58

two main species: Alcanivorax borkumensis Oleispira antarctica can break down hydrocarbons to CO2 and energy however cannot break down polyaromatic hydrocarbons as complex structure require specific O2, nitrogen, phosphorus and temp to live and often this not correct in oil spill zones of ocean

Question 59

describe Alcanivorax borkumensis?

Answer 59

aerobic hydrcarbonoclastic bacterium which propagates in seawater containing crude oil oil leakage increases phosphorous and nitrogen which is natural nutrient for bacteria = better growth produces biosurfactants which break down water surface tension so oil forms droplet bacterial then covers droplets w biofilm

Question 60

describe Oleispira antarctica?

Answer 60

aerobic/anaerobic hydrocarbonoclastic bacterium which lives in cold marine water can degrade oil in cold and deep water growth temp. between 1-15 degrees celsius

Question 61

what are some examples of fungi we could use for bioremediation?

Answer 61

oyster mushroom - eats diesel and other petroleum products producing CO2 and H2O as result white rot fungus - can break down polyaromatic hydrocarbons that the bacteria couldnt prod H2O2

Question 62

what are some of the drawbacks of using fungi for bioremediation?

Answer 62

they grow slow af so slow process they will also degrade biological material so other environmental consequences (this one applies to bacteria too)

Question 63

what are the two types of bioremediation?

Answer 63

biostimulation - environmental modification by adding nutrients to stimulate certain microbes or aerating soil/water bioaugmentation - addition of microbes

Question 64

what are the advantages of biostimulation?

Answer 64

cheaper natural process so publicly acceptable less disturbing to enviro destroys wide range of contaminants

Question 65

what are the disadvantages of biostimulation?

Answer 65

often unpredictable or unsuccessful physical environment challenging due to pH, temp, O2 etc. (so adding nutrients to certain enviro may not help growth as other factors limiting) concentration dependent slow process biological competition

Question 66

what are some problems with bioaugmentation?

Answer 66

bacteria require certain nutrients that might not be present not enough bacterial numbers and they just get diluted in enviro could disturb ecological niches

Question 67

what is bioaugmentation good for?

Answer 67

degrading specific soil or groundwater contaminants such as chlorinated solvents or petroleum

Question 68

describe the Pseudomonas species?

Answer 68

rod-shaped bacterial species opportunistic pathogen some can protect crops from pests and disease some can cause precipitation by cooling vapour and some can utilise petroleum products so can be used to clean up spills

Question 69

what is biodegradation?

Answer 69

natural break down of everything which replenishes ecosystem microbes degrade organic matter slowly

Question 70

what are the stages of biodegradation?

Answer 70

1. biodeteriation - surface level degradation where material properties modified 2. biofragmentation - degrades polymers to oligomers and monomers; if anaerobic prod methane, if aerobic prod CO2 and H2O 3. assimilation/mineralisation - microbes use the products of prev step

Question 71

what are some common plastic degrading microbes?

Answer 71

Penicillum, Aspergillus, Pseudomonas sp. these can degrade non-biodegradable plastics and consume for growth by secreting PETase enzymes slow degradation time so no commercial scale also some gut microbes and also mealworms

Question 72

what are xenobiotics?

Answer 72

foreign compounds introduced into environment by humans

Question 73

give an example of a xenobiotic and how it is problematic?

Answer 73

DDT is a pesticide which kills insects and thus can control malaria and typhus outbreaks still present in soil, has detrimental reproductive impacts (oestrogen blocker) and causes cancer

Question 74

why are xenobiotics usually so hard to get rid of?

Answer 74

they usually stable for a long time have complex ring structures and stable C-halogen bonds which are highly chlorinated very recalcitrant to microbial attack shortage of enzymes to get rid of them

Question 75

how can we degrade DDT?

Answer 75

reduce the organochlorines (used in pesticides) DDT converted to DDE and then to DDD this can be done with sulcate-reducing bacterium such as Clostridium sp.

Question 76

what is PCB?

Answer 76

a very chlorinated and very recalcitrant xenobiotic used in coolant fluids it may not degrade at all takes a long time to test for biodegradation

Question 77

what happens to waste material from an organism in the environment?

Answer 77

either used directly by another organism or converted

Question 78

what are common liquid wastes humans produce?

Answer 78

shower water urine washing-machine water trade wastes and other industrial wastes

Question 79

what are some solid wastes humans produce?

Answer 79

human faeces kitchen waste (nowhere near as much liquid waste and gets mixed w water for transport e.g. flush toilet)

Question 80

what is mixed domestic sewage?

Answer 80

our combined liquid and solid wastes

Question 81

what does waste water include and what are there in high levels?

Answer 81

includes sewage, industrial and agricultural effluent, street runoff collected in drains contains high levels of organic matter, heavy metals, nutrients and microorganisms

Question 82

what are some common diseases that are spread in wastewater?

Answer 82

typhoid cholera COVID-19 (not really common but relevant)

Question 83

what are the main microbes of human origin in wastewater?

Answer 83

coliforms such as E. coli presence of E. coli indicates faecal matter from warm-blooded animals (hence use as indicator bacteria) some disease causing pathogens e.g. Salmonella can spread and re-enter human food chain e.g. through shellfish can contaminate groundwater

Question 84

what are the goals of wastewater treatment?

Answer 84

eradicate any human pathogen seperate wastewater into sludge and a dissolved fraction containing water, organic material, bacteria and salts reduce organic load (microbes, insoluble debri, soluble organic matter) remove chemical compounds e.g. N and P make it safe for marine disposal

Question 85

what occurs in primary treatment of wastewater?

Answer 85

wastewater enters treatment plant passed through milliscreens which sieve for large solids (anything that doesn't go through goes to landfill) then through grit chambers which slow the flow and into primary clarifier

Question 86

what does the primary clarifier do?

Answer 86

separates suspended solids from liquid gravity pulls smaller particles into sludge and lighter solids float at top and accumulate as scum sludge goes into waste disposal area

Question 87

what occurs in secondary treatment of wastewater?

Answer 87

aeration basins blow air in to keep mixture in motion and provides oxygen environmentally friendly bacteria (large consortia) feed on nutrients in wastewater e.g. fats, sugar. ammonia bacterial clusters form as they break down waste; can form biofilms which are removed via settlement secondary clarifier separates bacterial clusters from liquid through flocculation activated sludge pumped back into aeration basin to provide more bacteria for breakdown separation of solids from liquids and then centrifugation (liquid back into aeration basin and sludge disposed)

Question 88

what occurs in final/tertiary treatment?

Answer 88

disinfection to kill any remaining microorganisms; traditionally used chlorine (effluent <0.5mg/L Cl) filtration (e.g. gravel or sand) UV light disinfection release into harbour

Question 89

how can we find microbes for remediation?

Answer 89

isolation and characterisation of microbes from unique habitats traditional lab conditions that test microbial consortia to perform bioremediation culture-based techniques (problematic cause so many microbes unculturable) DNA-based techniques are what we now turning towards

Question 90

what are some DNA-based techniques for identification of microorganisms?

Answer 90

16S rRNA sequencing Omics-approaches

Question 91

what are the omics-approaches?

Answer 91

discover novel microbes not accessible w traditional culturing explore metagenomes of contaminated environmental samples for their microbial communities (gives insight on diversity) allows to design and develop efficient strains of microbes e.g. for better metabolism of xenobiotics

Question 92

what is Pseudomonas putida?

Answer 92

soil bacterium and plant coloniser adapted lifestyle to harsh environmental conditions and stresses remarkable metabolic and physiological robustness uses wide variety of C and N sources can use omics to engineer new optimised strains

Question 93

what is metabolic engineering?

Answer 93

purposeful modification of cellular networks including metabolic, gene regulatory, and signalling networks to achieve desirable goals e.g. enhanced production of metabolites, increase intracellular levels of essential precursors, nutrient source uptake and by-product formation

Question 94

how can metabolically engineer Pseudomonas putida for better bioremediation?

Answer 94

synthetic biology tools combined with omics data can be used to re-purpose the central carbon metabolism leading to synthesis of new chemical structures

Question 95

what are some problems with bioengineering and how can we overcome them?

Answer 95

production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing overcome by restructuring existing pathways and developing new ones with different enzymes

Question 95

what are some problems with bioengineering and how can we overcome them?

Answer 95

production of dead-end substrates e.g. reactive intermediates which cannot be metabolised so bacteria stop growing overcome by restructuring existing pathways and developing new ones with different enzymes

Question 96

what are DCA and TCP examples of?

Answer 96

carcinogenic and mutagenic xenobiotics which cause reproductive effects highly recalcitrant in enviro and initially used as degreasing agent spread via groundwater flows

Question 97

how can bacterial degradation of DCA occur and how could we use this for large scale industrial applications?

Answer 97

Xanthobacteri autotrophicus pathway can degrade it by: haloalkane dehalogenase hydrolyses one C-Cl bond dehydrogenase produces chloroacetic acid dehalogenase converts this to glycolic acid which then can turn to CO2 if we introduce this pathway into P. putida could be used for large scale applications

Question 98

how can bacterial degradation of TCP occur?

Answer 98

natural organisms cannot mineralise TCP, however similarly structured compounds are biodegradable protein and metabolic engineering to construct microbes with improved catabolic activities

Question 99

how could we engineer DCP degrading enzymes to degrade TCP?

Answer 99

haloalkane dehalogenase (DhIA) catalyses first step in DCA degradation and could be engineered to have wider substrate range this engineering could be done through site-directed mutagenesis to improve activity or modification of tunnel proteins

Answer 100

very strict here but some techniques no approved e.g. more protein-protein expression which is way more safe and cost effective than other methods